Microbial-based waste water treatment compositions and methods of use thereof

ABSTRACT

The present invention relates to microbial compositions useful in treating and remediating wastewater, removing organic matter from the surfaces of post harvested fruits and vegetables, and decreasing post-harvest disease in fruit and vegetables.

RELATED APPLICATIONS

This application claims priority to and benefit of provisionalapplication U.S. Ser. No. 61/825,332 filed on May 20, 2013, the contentsof which are herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to wastewater treatment compositionscontaining micro-organisms and methods of using the compositions.

BACKGROUND OF THE INVENTION

Various sewage treatment methods and plants are known. Most largemunicipal systems employ a series of settling ponds sequentiallyconcentrating the solids contained in wastewater either with or withoutpolymers for separation from liquids via mechanical separation means,such as belt presses. In order to produce a clean effluent that can besafely discharged to watercourses, wastewater treatment operations usethree or four distinct stages of treatment to remove harmfulcontaminants.

Preliminary wastewater treatment usually involves gravity sedimentationof screened wastewater to remove settled solids. Half of the solidssuspended in wastewater are removed through primary treatment. Theresidual material from this process is a concentrated suspension calledprimary sludge, which will undergo further treatment to becomebiosolids.

Secondary wastewater treatment is accomplished through a biologicalprocess, which removes biodegradable material. This treatment processuses microorganisms to consume dissolved and suspended organic matter,producing carbon dioxide and other by-products. The organic matter alsoprovides nutrients needed to sustain the communities of microorganisms.As microorganisms feed, their density increases and they settle to thebottom of processing tanks, separated from the clarified water as aconcentrated suspension called secondary sludge, biological sludge,waste activated sludge, or trickling filter humus.

Tertiary or advanced treatment is used when extremely high-qualityeffluent is required, such as direct discharge to a drinking watersource. The solid residual collected through tertiary treatment consistsmainly of chemicals added to clean the final effluent, which arereclaimed before discharge, and therefore not incorporated intobiosolids.

What is needed are compositions and methods that can performbioremediation, to remediate materials such as water that have an excessof unwanted biological material or other contaminating or pollutingcompounds,.

SUMMARY OF THE INVENTION

In various aspects the invention provides compositions containing amixture of micro-organisms for degrading organic matter in augmentingthe treatment of wastewater. Importantly, the compositions of theinvention fully disperse in water and does not require a preactivationof the bacteria prior to use.

In various aspects the invention provides compositions for degradingorganic matter. The compositions contain a mixture of Bacillus organismsor a mixture of Bacillus and Lactobacillus. In some embodiments thecompositions contain Bacillus subtilis, Bacillus amyloliquefaciens,Bacillus licheniformis, Bacillus pumilus, Pediococcus acidilactici,Pediococcus pentosaceus and Lactobacillus plantarum. In otherembodiments the composition contains Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillusmeagerium, Bacillus coagulans, and Paenibacillus polymyxa. Each of theorganisms in the mixture is individually aerobically (Bacillus) oranaerobically (Lactobacillus) fermented, harvested, dried, and ground toproduce a powder having a mean particle size of about 200 microns, withgreater than about 60% of the mixture in the size range between 100-800microns. In some embodiments, the ratio of the Bacillus to Lactobacillusis between 1:10 to 10:1. Preferably, the ratio of the Bacillus toLactobacillusis 1:10 Bacillus to Lactobacillus. In other embodimentsthethe ratio of the Lactobacillus is 1:1:1.

In some aspects the composition has a moisture content of less thanabout 5%; and a final bacterial concentration of about between 10⁵-10¹¹colony forming units (CFU) per gram of the composition.

In various aspects the composition further contains an inert carriersuch as dextrose monohydrate. Preferably, the dextrose monohydrate is ata concentration of about between 75-95% (w/w).

In other aspects the composition further includes an organic emulsifier.The organic emulsifier is for example, soy lecithin. Preferably, theorganic emulsifier is at a concentration of about between 2 to 5% (w/w).

Also included in the invention are methods treating wastewater bycontacting the wastewater with a composition containing the Bacillus andLactobacillus mixtures of the invention. The wastewater is for example,municipal sewage, residential septic, or industrial wastewater. Thewastewater contains food, fats, oils, grease, brewery, agriculture, orcommodity waste. The method results in decreasing biological oxygendemand (BOD), total suspended solids (TSS) total kjeldahll nitrogen(TKN) and fats, oils and grease (FOG) in the wastewater.

Further included in the invention are methods of treating swimming poolwater comprising contacting the water by contacting the wastewater witha composition containing the Bacillus and Lactobacillus mixtures of theinvention. In some aspects the water is contacted by contacting aswimming pool filtration unit with the composition. In other aspects thecomposition is imbedded in a solid support.

In yet another aspect the invention provides methods of cleaningartificial turf, by contacting the artificial turf with a compositioncontaining the Bacillus and Lactobacillus mixtures of the invention.

In another aspect the invention includes methods of remediatingwastewater from fruit or vegetable by contacting the waste water withthe Bacillus mixtures of the invention.

In a further aspect the invention provides methods of removing organicmatter from the surfaces of fruits and vegetables by contacting thefruit or vegetable with the Bacillus mixtures of the invention. Themethod improves the shelf life and/or appearance, of the fruit orvegetables. The fruit is for example, a banana.

Also provided by the invention are methods of manufacturing thecompositions of the invention. Mixtures of bacteria containing Bacillusand Lactobacillus, are manufactured by individually aerobicallyfermenting each Bacillus organism; individually anaerobically fermentingeach Lactobacillus organism; harvesting each Bacillus and Lactobacillusorganism; drying the harvested organisms;grinding the dried organisms toproduce a powder combining each of the Bacillus powders to produce aBacillus mixture; combining each of the Lactobacillus powders in equalamounts to produce a Lactobacillus mixture and combining the Bacillusmixture and the Lactobacillus mixture at a ratio of between 1:10 to10:1. The Bacillus organisms are Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, and Bacillus pumilus. TheLactobacillus comprises Pediococcus acidilactici, Pediococcuspentosaceus and Lactobacillus plantarum, Bacillus meagerium, Bacilluscoagulans, and Paenibacillus polymyxa. The mixture has a moisturecontent of less than about 5%; and a final bacterial concentration ofbetween about 10⁵-10¹¹ colony forming units (CFU) per gram of thecomposition.

Mixtures of bacteria containing Bacillus organisms are manufactured byindividually aerobically fermenting Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillusmeagerium, Bacillus coagulans, and Paenibacillus polymyxa; harvestingeach organism, drying the harvested organisms; grinding the driedorganisms to produce a powder and combining each of the Bacilluspowders. The mixture has a moisture content of less than about 5%; and afinal bacterial concentration of between about 10⁵-10¹¹ colony formingunits (CFU) per gram of the composition.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Other features and advantages of the invention will be apparent from andencompassed by the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows results of BOD testing of the microbial compositions of theinvention

FIG. 2: shows results of BOD Reduction in Residential LPP Systems

FIG. 3: shows the FOG Reduction in Residential LPP Systems

FIG. 4: shows the TSS Reduction in Residential LPP Systems

FIG. 5 is a photograph showing the reduction of turbidity of banana washwater upon treatment with the compositions of the invention.

FIG. 6 are photographs showing bananas treated conventionally (A) andwith the compositions of the invention (B).is a photograph showing thereducing of turbidity of banana wash water upon treatment with thecompositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides microbial compositions for augmenting wastewatertreatment and remediation. In some aspects, the microbial compositionscontain a mixture of Bacillus and Lactobacillus bacteria wherein theratio of bacillus to lactobacillus ranges from 1:10 to 10:1. In otheraspect the microbial composition contains a mixture of Bacillus.Specifically, the Bacillus and Lactobacillus compositions comprisemixtures of Bacillus subtilis, Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus pumilus, Pediococcus acidilactici, Pediococcuspentosaceus and Lactobacillus plantarum The Bacillus compositionscomprise mixtures of Bacillus subtilis, Bacillus amyloliquefaciens,Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillusmegaterium, and Paenibacillus polymyxa.

Importantly, the composition fully disperses upon the addition to waterand unlike other wastewater treatment microbial compositions thecompositions does not require a preactivation of the bacteria prior touse.

The microbial compositions reduce biological oxygen demand (BOD), totalsuspended solids (TSS) total kjeldahll nitrogen (TKN) and fats, oils andgrease (FOG) in wastewater. The compositions are also used to degradelatexes enabling their removal from waters used to wash bananas, orother fruits and vegetables and to remove accumulating scum and algaefrom swimming pools. Additionally, it was surprisingly discovered thatthe compositions not only degrade latexes from the wastewaters resultingfrom the washing of bananas, or other fruits and vegetables, but alsoreduced the incidence of post-harvest disease in the banana harvest.

The term “microbial, bacteria” or “microbes” as used herein, refers tomicroorganisms that confer a benefit. The microbes according to theinvention may be viable or non-viable. The non-viable microbes aremetabolically-active. By “metabolically-active” is meant that theyexhibit at least some residual enzyme, or secondary metabolite activitycharacteristic to that type of microbe.

By the term “non-viable” as used herein is meant a population ofbacteria that is not capable of replicating under any known conditions.However, it is to be understood that due to normal biological variationsin a population, a small percentage of the population (i.e. 5% or less)may still be viable and thus capable of replication under suitablegrowing conditions in a population which is otherwise defined asnon-viable.

By the term “viable bacteria” as used herein is meant a population ofbacteria that is capable of replicating under suitable conditions underwhich replication is possible. A population of bacteria that does notfulfill the definition of “non-viable” (as given above) is considered tobe “viable”.

“Waste holding facility” as used herein is meant a facility for theholding, storage, and treatment of organic wastes.

“Wastewater,” as used herein, is principally directed to domestic sewagefrom dwellings, business buildings, institutions, which contain groundwater, surface water, and/or storm water. Wastewater also includes wateras a result of processing, or washing of products such as fruit andvegetables. For the purposes of this invention, swimming pool water isincluded in the definition of “wastewater”.

“Treating” as used herein is means inoculating organic waste withmicrobes designed to enhance efficient degradation of organic matter.

Unless stated otherwise, all percentages mentioned in this document areby weight based on the total weight of the composition.

The microbes used in the product according to the present invention maybe any conventional mesophilic bacteria. It is preferred that thebacteria are selected from the Lactobacillacae and Bacillaceae families.More preferably the bacteria selected form the genus Bacillus andLactobacillus are included in the compositions of the invention.

Preferred are compositions wherein the ratio of the Bacillus toLactobacillus is between 1:10 to 10:1. Preferably, the ratio of theBacillus to Lactobacillus is 1:10.

Other preferred compositions are wherein the ratio of the Lactobacillusis preferablyl 1:1:1.

The levels of the bacteria to be used according the present inventionwill depend upon the types thereof. It is preferred that the presentproduct contains bacteria in an amount between about 10⁵ and 10¹¹ colonyforming units per gram.

The bacteria according to the invention may be produced using anystandard fermentation process known in the art. For example, solidsubstrate or submerged liquid fermentation. The fermented cultures canbe mixed cultures or single isolates.

In some embodiments the bacteria are anaerobically fermented in thepresence of carbohydrates. Suitable carbohydrates include inulin,fructo-oligosaccharide, and gluco-oligosaccharides.

The bacterial compositions are in powdered, dried form. Alternatively,the bacterial compositions are in liquid form.

After fermentation the bacteria are harvested by any known methods inthe art. For example the bacteria are harvested by filtration orcentrifugation.

The bacteria are dried by any method known in the art. For example thebacteria are air dried, or dried by freezing in liquid nitrogen followedby lyophilization.

The compositions according to the invention have been dried to moisturecontent less than 20%, 15%, 10% 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.Preferably, the composition according to the invention has been dried tomoisture content less than 5%.

In some embodiments the dried powder is ground to decrease the particlesize. The bacteria are ground by conical grinding at a temperature lessthan 10 ° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° C., 1° C., 0°C., or less. Preferably the temperature is less than 4° C.

For example the particle size is less than 1500, 1400, 1300, 1200, 1100,1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 microns.Preferably, the freeze dried powder is ground to decrease the particlesize such that the particle size is less than 800 microns. Mostpreferred are particle sizes less than about 400 microns. In mostpreferred embodiments, the dried powder has a mean particle size of 200microns, with 60% of the mixture in the size range between 100-800microns. In various embodiments the freeze dried powder is homogenized.

In various embodiments the bacteria compositions are mixed with an inertcarrier such as dextrose monohydrate. The dextrose monohydrate is at aconcentration of at least 60%, 70%, 75%, 80%, 85%, 90%, 95% or more.Preferably, the dextrose monohydrate is at a concentration of aboutbetween 75-95% (w/w).

In other aspects the bacterial compositions contain an organicemulsifier such as, for example, soy lecithin. The organic emulsifier isat a concentration of about 1%, 2%, 3%, 4%, 5%, 5, 7%, 8%, 9% or 10%.Preferably, the organic emulsifier is at a concentration of between 2 to5% (w/w).

Further, if desired, the bacterial compositions may be encapsulated tofurther increase the probability of survival; for example in a sugarmatrix, fat matrix or polysaccharide matrix.

The bacterial compositions of the invention are used to treatcommercial, municipal, industrial, and residential wastewater, includingwater in commercial, municipal and residential swimming pools. Thebacterial compositions can also be used to remove organic matter from,artificial grass surfaces, such as, for example, used Astroturf.

One or more embodiments relate generally to wastewater treatmentmethods. A waste treatment system may receive wastewater from acommunity, industrial, or residential source during typical operation.For example, the wastewater may be delivered from a municipal or otherlarge-scale sewage system. Alternatively, the wastewater may begenerated, for example, by food processing or pulp and paper plants.

Wastewater may generally be any stream of waste, bearing at least oneundesirable organic constituent. The waste products treatable with thepresent invention include, but are not limited to organic waste producedby metabolic processes, including human and animal waste, as well asindustrial wastes, effluents, sewage, and the like.

The aqueous solution or the dry composition according to the inventioncan be employed to reduce biological oxygen demand (BOD), totalsuspended solids (TSS) total kjeldahll nitrogen (TKN) and fats, oils andgrease (FOG) in sewage and other waste water products. The compositionsof the invention may also be used to treat swimming pools to remove scumand reduce algae.

The compositions biodegrades latex from waters used to wash bananas. Thecompositions of the invention are also useful in removing organicmaterial from the surfaces of fruits and vegetables, such as, forexample, in the processing or washing of fruit and vegetables, e.g.,which aids in improving fruit shelf life, appearance, and reducing theincidence of post-harvest disease.

Solutions of the compositions can be pumped into the material to betreated (liquid, sludge, or solid) or sprayed onto the surface, or intothe airspace surrounding the material, or applied to a filter throughwhich the water to be cleaned is passed. The dry material can be mixedinto a slurry or solution at the point of application and applied in asimilar manner.

The compositions or the invention are manufactured by any methodsuitable or productions of bacterial compositions. Preferably, mixturesof bacteria containing Bacillus and Lactobacillus, are manufactured byindividually aerobically fermenting each Bacillus organism; individuallyanaerobically fermenting each Lactobacillus organism; harvesting eachBacillus and Lactobacillus organism; drying the harvested organisms;grinding the dried organisms to produce a powder combining each of theBacillus powders to produce a Bacillus mixture; combining each of theLactobacillus powders in equal amounts to produce a Lactobacillusmixture and combining the Bacillus mixture and the Lactobacillus mixtureat a ratio of between 1:10 to 10:1. The Bacillus organisms are Bacillussubtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, andBacillus pumilus. The Lactobacillus comprises Pediococcus acidilactici,Pediococcus pentosaceus and Lactobacillus plantarum, Bacillus meagerium,Bacillus coagulans, and Paenibacillus polymyxa. The mixture has amoisture content of less than about 5%; and a final bacterialconcentration of between about 10⁵-10¹¹ colony forming units (CFU) pergram of the composition.

Mixtures of bacteria containing Bacillus organisms are manufactured byindividually aerobically fermenting Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillusmeagerium, Bacillus coagulans, and Paenibacillus polymyxa; harvestingeach organism, drying the harvested organisms; grinding the driedorganisms to produce a powder and combining each of the Bacilluspowders. The mixture has a moisture content of less than about 5%; and afinal bacterial concentration of between about 10⁵-10¹¹ colony formingunits (CFU) per gram of the composition.

A better understanding of the present invention may be given with thefollowing examples which are set forth to illustrate, but are not to beconstrued to limit the present invention.

EXAMPLES Example 1 Preparation of the Microbial Species

The microbes of the present invention are grown using standard deep tanksubmerged fermentation processes known in the art.

Bacillus Species

Individual starter cultures of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, and Bacillus pumilus aregrown according to the following general protocol: 2 grams NutrientBroth, 2 grams AmberFerm (yeast extract) and 4 grams Maltodextrin areadded to a 250 ml Erlenmeyer flask. 100 mls distilled, deionized wateris added and the flask is stirred until all dry ingredients aredissolved. The flask is covered and placed for 30 min in an Autoclaveoperating at 121° C. and 15 psi. After cooling, the flask is inoculatedwith lml of one of the pure microbial strains. The flask is sealed andplaced on an orbital shaker at 30° C. Cultures are allowed to grow for3-5 days. This process is repeated for each of the microbes in themixture. In this way starter cultures of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, and Bacillus pumilus areprepared.

Larger cultures are prepared by adding 18 grams Nutrient Broth, 18 gramsAmberFerm, and 36 grams Maltodextrin to 1 liter flasks with 900 mlsdistilled, deionized water. The flasks are sealed and sterilized asabove. After cooling, 100 mls of the microbial media from the 250 mlErlenmeyer flasks are added. The 1 liter flasks are sealed, placed onand orbital shaker, and allowed to grow out for another 3-5 days at 30°C.

In the final grow-out phase before introduction to the fermenter, thecultures from the 1 liter flasks are transferred under sterileconditions to sterilized 6 liter vessels and fermentation continued at30° C. with aeration until stationary phase is achieved. The contents ofeach 6 liter culture flask are transferred to individual fermenterswhich are also charged with a sterilized growth media made from 1 partyeast extract and 2 parts dextrose. The individual fermenters are rununder aerobic conditions at pH 7.0 and the temperature optimum for eachspecies:

Temperature Microbe Optimum Bacillus subtilis 35° C. Bacillusamyloliquefaciens 30° C. Bacillus licheniformis 37° C. Bacillus pumilus32° C.

Each fermenter is run until cell density reaches 10¹¹ CFU/ml, onaverage. The individual fermenters are then emptied, filtered, andcentrifuged to obtain the bacterial cell mass which is subsequentlydried under vacuum until moisture levels drop below 5%. The finalmicrobial count of the dried samples is 10¹⁰-10¹¹ CFU/g.

Lactobacillus Species

Individual, purified isolates of Pediococcus acidilactici, Pediococcuspentosaceus and Lactobacillus plantarum are grown-up in separatefermenters using standard anaerobic submerged liquid fermentationprotocols at the pH and temperature optimum for each species:

pH Temperature Microbe Optimum Optimum Pediococcus acidilactici 5.5 37°C. Pediococcus pentosaceus 5.5 37° C. Lactobacillus plantarum 6.0 35° C.

After fermentation the individual cultures are filtered, centrifuged,freeze dried to a moisture level less than about 5%, then ground to aparticle size of about 200 microns.

The dried bacillus and lactobacillus microbes are combined to give afinal dried microbial composition comprising Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniforms, Bacillus pumilus, Pediococcusacidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum, at aratio of total bacillus to lactobacillus between 1:10 and 10:1 with amicrobial activity between 10⁸ and 10¹° CFU/g.

Example 2 Preparation of the Microbial Species Via Solid SubstrateFermentation

The microbial mix of the present invention can also be prepared viasolid substrate fermentation according to the following process:

Bacillus Species

Four pounds of Dairy 12% Mineral Mix, 60 lbs Rice bran, and 30 lbsSoybean meal were added to a jacketed, horizontal mixer with screwauger. Water and steam were added with mixing to obtain slurry. Aftermixing for 2 minutes, 300 lbs wheat bran was added to the mixer followedby more water and steam to re-make the slurry. With the mixertemperature controlled to 35-36° C., 4 lbs of a dry microbial mixturecomprising Bacillus subtilis, Bacillus amyloliquefaciens, Bacilluslicheniformis and Bacillus pumilus with an initial microbial activity ofabout 1×10¹° CFU/g, were added. The mixer was closed; temperatureadjusted to 34° C., and the contents allowed to mix for up to 4 days.After fermentation the contents of the mixer were emptied onto metaltrays and allowed to air dry. After drying, a product was ground to aparticle size below about 200 microns. The final bacillus productobtained had a microbial count on the order of 1×10¹¹ CFU/g and lessthan about 5% moisture.

Lactobacillus Species

A mixed culture of Pediococcus acidilactici, Pediococcus pentosaceus andLactobacillus plantarum was fermented under GMP conditions for up to 5days on a mixture comprised of: 1 part inulin, 2.2 parts isolated soyprotein, 8 parts rice flour with 0.25% w/w sodium chloride, 0.045% w/wCalcium carbonate, 0.025% w/w Magnesium sulphate, 0.025% w/w Sodiumphosphate, 0.012% w/w Ferrous sulphate and 29.6% water. Upon completionof fermentation the mixture was freeze dried to a moisture content lessthan 5%, ground to a particle size below 800 microns and homogenized.The final microbial concentration of the powdered product is between 10⁹and 10¹¹ CFU/g.

Final Microbial Mix

The dried bacillus and lactobacillus microbes were combined in ratiosbetween 1:10 and 10:1 to give a final dried microbial compositioncomprising Bacillus subtilis, Bacillus amyloliquefaciens, Bacilluslicheniformis, Pediococcus acidilactici, Pediococcus pentosaceus, andLactobacillus plantarum with a microbial activity between 10⁸ and 10¹⁰CFU/g.

Example 3 Identification of Optimum Bacillus and Lactobacillus Ratiosfor BOD Reduction

Two microbial compositions were prepared by mixing the individual driedmicrobes (bacillus and lactobacillus) from Example 1 in a ratio of 1:1(Composition A) and 1:10 (Composition B). These two compositions werecompared for their ability to reduce BOD of dairy lagoon wastewater(FIG. 1). A commercial wastewater product (BiOWiSH Aqua, made inThailand) was included as a positive control These results show that the1:10 ratio of Bacillus to Lactobacillus (Composition B) is preferred forrapid reduction of BOD.

Example 4 Formulation of the Wastewater Product using Microbes fromExample 1

The individual dried microbes (bacillus and lactobacillus) from Example1 were mixed together in the ratio 1:10 (Bacillus:Lactobacillus). Thisdried microbial mix was diluted 1:100 with dextrose (Clintose®Industrial Dextrose). To this mix was added 3% by weight of powdered soylecithin (Nealanders International, Inc.). The final microbal count ofthis composition was 1×10⁸ CFU/g.

Example 5 Formulation of Wastewater Product using Process From Example 2

The bacillus and lactobacillus solid substrate fermentation productsfrom Example 2 were ground to about 200 micron average particle size,mixed together in equal proportion then mixed with 3% by weight powderedsoy lecithin (Nealanders International, Inc). A final product wasobtained with a microbial count of 10⁸-10⁹ CFU/g and moisture below 5%.

Example 6 Performance of the Waste Water Product from Example 4 inSeptic Treatment

Three residential Low Pressure Pipe (LPP) septic systems located incentral North Carolina, USA, were selected for testing. Baselinedeterminations of BOD (Biological Oxygen Demand), TSS (Total SuspendedSolids), and FOG (Fats Oil and Grease) were made then 200 g of theWastewater Treatment formulation from Example 4 were added to eachsystem every week for a period up to 8 weeks. Weekly recordings of BOD,TSS, and FOG were made for each system. Results, averaged across the 3LPP septic systems, showed significant reduction in the key biochemicalmeasures versus baseline for all three systems (FIGS. 2-4).

Example 7 Performance of the Waste Water Product from Example 5 inSeptic Treatment

Three residential LPP septic systems in central North Carolina, USA,were selected for this study. A baseline determination of BOD, TSS, TKN,and FOG was made for each system then 200 grams of the composition fromExample 5 were added to each system every week for a period up to 8weeks. BOD, TSS, TKN, and FOG were recorded weekly for each of the threesystems. Results, averaged across the 3 LPP septic systems, showsignificant reduction in the key biochemical measures versus baselinefor all three systems:

BOD (mg/l) % Reduction Treatment Baseline Day 7 Day 14 Day 28 Day 56 vs.Baseline System US EPA Regulation 350 350 350 350 350 level LPP 200 gweekly dosage 1636 564 293 236 189 88.5 Avg. of the wastewatercomposition from Example 5

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TSS (mg/l) % Reduction Treatment Baseline Day 7 Day 14 Day 28 Day 56 vs.Baseline System US EPA Regulation 100 100 100 100 100 Level LPP 200 gweekly dosage 622.6 345.8 196 63.4 58.7 90.6 Avg. of the wastewatercomposition from Example 5

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TKN (mg/l) % Reduction Treatment Baseline Day 7 Day 14 Day 28 Day 56 vs.Baseline System US EPA Regulation 100 100 100 100 100 Level LPP 200 gweekly dosage 103.8 50.3 62.7 42.8 57.8 44.4 Avg. of the wastewatercomposition from Example 5

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FOG (mg/l) % Reduction Treatment Baseline Day 7 Day 14 Day 28 Day 56 vs.Baseline System US EPA Regulation 30 30 30 30 Level LPP 200 g weeklydosage 280.3 104.7 46.7 34.2 111 60.4 Avg. of the wastewater compositionfrom Example 5

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Example 8 Reduction in BOD Compared to Competitive Product

Waste water was collected from local dairy lagoons in California'sCentral Valley area. Samples were pooled to create a common stocksolution. The stock waste water was pipetted into several 300 ml BODbottles and the following experimental design set-up:

Bottle Treatment 1 Control A (no added microbes) 2 Control B (no addedmicrobes)  3-10 100 ppm of the waste water treatment from Example 4.11-13 100 ppm of a commercial, mixed microbe waste water treatmentproduct (BiOWiSH ™ Aqua FOG)

Initial BOD was measured for each Bottle then again after storage at 30°C. for 5 days. On average, the control showed a BOD reduction of 669mg/l versus 742 mg/l for the wastewater treatment composition fromExample 4 and 701 mg/l for the commercial product.

Avg. BOD Avg. BOD BOD₅ Initial BOD₅ Final reduction reduction vs. Bottle(mg/l) (mg/l) (mg/l) Control (mg/l) 1 825 117 669 — 2 813 183 3 900 128742 10.9% 4 926 238 5 914 120 6 836 89 7 886 102 8 870 131 9 930 302 10887 102 11 842 118 701 4.8% 12 812 139 13 879 174

Example 9 Reduction in Total Suspended Solids Versus Competitive Product

Waste water was collected from the dairy lagoon pond at CaliforniaPolytechnic State University in San Luis Obispo, Calif., distributedamong several 2 liter bottles and the following experimental designset-up:

Bottle Treatment 1 Control (no added microbes) 2-4 100 ppm of the wastewater treatment from Example 5. 5 100 ppm of a commercial, mixed microbewaste water treatment product (BiOWiSH ™ Aqua FOG)

The bottles were kept at 30° C. for 5 weeks. Every week, two 50 mlaliquots were collected and dried at 90° C. for Total Solidsdetermination:

TSS (mg/l) % Final TSS Week 0 1 2 3 4 5 Reduction Bottle 1 1819.7 1026.7933.3 600.0 453.3 386.7 78.8 (Control) Average Bottle 2 1750.0 866.7626.7 413.3 453.3 200.0 88.6 Average Bottle 3 1916.7 1080.0 720.0 640.0480.0 80.0 95.8 Average Bottle 4 2125.0 1173.3 786.7 546.7 133.3 93.395.6 Average Bottle 5 1708.3 1120.0 813.3 666.7 333.3 346.7 79.7 Average

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The bottles containing the wastewater treatment from Example 5 showedhigher overall percent TSS reduction than both the control and thecommercial product.

Example 10 Comparison Versus Competitive Septic Treatment System

Several residential LPP septic systems in central North Carolina, USA,were selected for this study. A baseline determination of BOD, TSS, TKN,and FOG was made for each system then each system was treated weeklyeither with 200 grams of the composition from Example 5 or 200 gram of acommercial product (BiOWiSHTM Aqua FOG). BOD, TSS, TKN, and FOG wererecorded weekly for each system. Results, averaged across the LPP septicsystems, are shown below:

(BOD (mg/l) % Reduction Treatment Baseline Day 7 Day 14 Day 28 Day 56vs. Baseline System US EPA Regulation 350 350 350 350 350 level Avg. ofLPP 200 g weekly dosage 1636 564 293 236 189 88.5 systems of thewastewater treated with composition from Example 5 Example 5 CompositionAvg. of LPP 200 g weekly dosage 2020 502 760 382 518 74.4 systems ofcommercial septic treated with treatment products Commercial Product

TSS (mg/l) % Reduction Treatment Baseline Day 7 Day 14 Day 28 Day 56 vs.Baseline System US EPA Regulation 100 100 100 100 100 Level Avg. of LPP200 g weekly dosage 622.6 345.8 196 63.4 58.7 90.6 systems of thewastewater treated with composition from Example 5 Example 5 CompositionAvg. of LPP 200 g weekly dosage 720 2540 413 652 123 98.3 systems ofcommercial septic treated with treatment products Commercial Product

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TKN (mg/l) % Reduction Treatment Baseline Day 7 Day 14 Day 28 Day 56 vs.Baseline System US EPA Regulation 100 100 100 100 100 Level Avg. of LPP200 g weekly dosage 103.8 50.3 62.7 42.8 57.8 44.4 systems of thewastewater treated with composition from Example 5 Example 5 CompositionAvg. of LPP 200 g weekly dosage 98 91 51 96 53 45.9 systems ofcommercial septic treated with treatment products Commercial Product

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FOG (mg/l) % Reduction Treatment Baseline Day 7 Day 14 Day 28 Day 56 vs.Baseline System US EPA Regulation 30 30 30 30 30 Level Avg. of LPP 200 gweekly dosage 280.3 104.7 46.7 34.2 111 60.4 systems of the wastewatertreated with composition from Example 5 Example 5 Composition Avg. ofLPP 200 g weekly dosage 113 160 251 49 69 38.9 systems of commercialseptic treated with treatment products Commercial Product

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Example 11 Remediation of Waste Water from Fruit Washing

The composition of Example 4 was used to treat the waste water frombanana washing. Latex fluid is released when bananas are harvested.Typically, the bananas are immersed in moving water to remove the latex.The waste water that results from this process is generally high inlatex concentration limiting the ability to recycle and reuse.

A test program was set-up in collaboration with Coorporación BananeraNacional (Costa Rica) to evaluate the ability of the waste watertreatment compositions of the present invention to remove latex andreduce incidence of post-harvest disease.

Samples were collected from waste water pools formed from washing pilesof bananas. The samples were collected late in the day when organic loadin the water was highest. Between 150 and 200 mls of waste water wereplaced in 250 ml Erlenmeyer flasks to which various concentrations ofthe waste water treatment composition from Example 4 were added.

The flasks were incubated for 12-72 hours at 24-26° C. with mildagitation (50 rpm on an orbital shaker). The microbial composition ofthe present invention was found to significantly reduce turbidity of thesolutions after 72 hours. (FIG. 5)

Based on these results a larger field trial was executed. On Day's 1 and2 of the field trial, banana wash water was treated with the dispersantBactrol® 500. 6.5 liters of Bactrol® were diluted into 60 liters ofwater and applied to bananas on the day of harvest via a drip systemplaced one meter above the banana stack. Chlorine was injected into thewater stream at a level of 2-3 mg/l throughout the day. On days 3 and 4of the test, the water was treated only with the waste water treatmentcomposition of Example 4 plus citric acid

Conventional Treatment DAY Bactrol ® 500 Chlorine Comments 1 6.5 Litersin 60 liters of Injected into the input pipe Water was not changed atthe water applied to the during the day to maintain a end of the day andwas used bananas during the day via concentration between 2-3 mg/l againthe next day drip 2 6.5 Liters in 60 liters of Injected into the inputpipe Water was discarded at the water applied to the during the day tomaintain a end of day two. bananas during the day via concentrationbetween 2-3 mg/l drip

Bioremediation Treatment Injection Citric Acid Cleaning Pool Drip TankTank (50 in Cleaning DAY (101m3) (50 liters) Liters pool Comments 3 101g of the All pools composition filled with from clean water to Example 4begin the next phase of the trial 4 150 g of the 150 g of the 1 kg (7am) composition composition 1 kg (10 am) from from 1 kg (1 pm) Example 4Example 4 (6:00 am) (6:00 am) + 50 g (1:00 pm) 5 150 g of the 150 g ofthe 1 kg (7 am) composition composition 1 kg (10 am) from from 1 kg (1pm) Example 4 Example 4 (6:00 am) (6:00 am) + 50 g (1:00 pm)

FIG. 6 compares bananas washed with the conventional treatment versusthose washed with the composition from Example 4.

Example 12 Swimming Pool Treatment

The microbial composition from Example 5 is dissolved in water at aconcentration of 100 g dried microbial product/liter. With the pump off,the microbial solution is poured into the filter unit of a residentialswimming pool having a noticeable scum layer on the surface and allowedto stand for 1 hour before the pump is turned-on. Within 24 hours thescum is significantly reduced and in 48 hours there is no visible scumremaining

Example 13 Expanded Microbial Composition for Wastewater Treatment andFruit/Vegetable Wash

A composition comprising the bacterial strains from Example 1 andadditional microbes selected for their ability to provide additionalwaste water treatment and fruit/vegetable wash benefits was designedusing a fermentation system similar to that developed in Example 1:

Bacillus and Paenibacillus species

Individual starter cultures of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus Pumilus, Bacilluscoagulans, Bacillus megaterium, and Paenibacillus polymyxa, were grownaccording to the following general protocol: 2 grams Nutrient Broth, 2grams AmberFerm (yeast extract) and 4 grams Maltodextrin were added to a250 ml Erlenmeyer flask. 100 milliters distilled, deionized water wereadded and the flask was stirred until all dry ingredients weredissolved. The flask was covered and placed for 30 min in an Autoclaveoperating at 121° C. and 15 psi. After cooling, the flask was inoculatedwith 1 ml of one of the pure microbial strains. The flask was sealed andplaced on an orbital shaker at 30° C. Cultures were allowed to grow for3-5 days. This process was repeated for each of the microorganism in themixture. In this way starter cultures of Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus Pumilus, Bacilluscoagulans, Bacillus megaterium, and Paenibacillus polymyxa wereprepared.

Larger cultures were prepared by adding 18 grams Nutrient Broth, 18grams AmberFerm, and 36 grams Maltodextrin to 1 liter flasks with 900mis distilled, deionized water.

The flasks were sealed and sterilized as above. After cooling, 100 misof the microbial media from the 250 ml Erlenmeyer flasks were added. The1 liter flasks were sealed, placed on and orbital shaker, and allowed togrow out for another 3-5 days at 30° C.

In the final grow-out phase before introduction to the fermenter, thecultures from the 1 liter flasks were transferred under sterileconditions to sterilized 6 liter vessels and fermentation continued at30° C. with aeration until stationary phase was reached. The contents ofeach 6 liter culture flask was transferred to individual fermenterswhich were also charged with a sterilized growth media made from 1 partyeast extract and 2 parts dextrose. The individual fermenters were rununder aerobic conditions at pH 7 at the temperature optimum for eachspecies:

Temperature Microbe Optimum Bacillus subtilis 35° C. Bacillusamyloliquefaciens 30° C. Bacillus licheniformis 37° C. Bacilluscoagulans 37° C. Bacillus megaterium 30° C. Bacillus pumilus 32° C.Paenibacillus polymyxa 30° C.

Each fermenter was run until cell density reached 10¹¹ CFU/ml, onaverage. The individual fermenters were then emptied, filtered, andcentrifuged to obtain the bacterial cell mass which was subsequentlydried under vacuum until moisture levels drop below 5%. The finalmicrobial count of the dried samples was 10¹⁰-10¹¹ CFU/g.

Lactobacillus Species

Individual, purified isolates of Pediococcus acidilactici, Pediococcuspentosaceus and Lactobacillus plantarum were grown-up in separatefermenters using standard anaerobic submerged liquid fermentationprotocols at the pH and temperature optimum for each species:

pH Temperature Microbe Optimum Optimum Pediococcus acidilactici 5.5 37°C. Pediococcus pentosaceus 5.5 37° C. Lactobacillus plantarum 6.0 35° C.

After fermentation the individual cultures were filtered, centrifuged,freeze dried to a moisture level less than about 5%, then ground to aparticle size of about 100 microns

The dried bacillus and lactobacillus microbes were combined in equalproportion to give a final dried microbial composition comprisingBacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis,Bacillus pumilus Bacillus coagulans, Bacillus megaterium, and,Paenibacillus polymyxa.

Example 14 Preparation of a Wastewater Product from the Expanded Set ofMicrobes in Example 13

The dried microbial mix of Example 13 is diluted 1:100 with dextrose(Clintose® Industrial Dextrose). To this mix is added 3% by weightpowdered soy lecithin (Nealanders International, Inc). The finalmicrobial count is typically 1×10⁹ CFU/g.

We claim:
 1. A composition for degrading organic matter, comprising amixture of: (a) Bacillus subtilis, Bacillus amyloliquefaciens, Bacilluslicheniformis, and Bacillus pumilus, wherein each of the Bacillus in themixture is individually aerobically fermented, harvested, dried, andground to produce a powder having a mean particle size of about 200microns, with greater than about 60% of the mixture in the size rangebetween 100-800 microns and either: (b) Pediococcus acidilactici,Pediococcus pentosaceus and Lactobacillus plantarum, wherein each of theLactobacillus in the mixture is individually anaerobically fermented,harvested, dried, and ground to produce a powder having a mean particlesize of about 200 microns, with greater than about 60% of the mixture inthe size range between 100-800 microns; or (c) Bacillus meagerium,Bacillus coagulans, and Paenibacillus polymyxa wherein each of theBacillus in the mixture are individually aerobically fermented ally,harvested, dried, and ground to produce a powder having a mean particlesize of about 200 microns, with greater than 60% of the mixture in thesize range between 100-800 microns wherein the composition upon additionto water fully disperses and does not require a preactivation of thebacteria.
 2. The composition of claim 1, wherein the ratio of theBacillus of step (a) to Lactobacillus of step (b) is between 1:10 to10:1.
 3. The composition of claim 2, wherein the ratio of the Bacillusof step (a) to Lactobacillus of step(b) is 1:10 Bacillus toLactobacillus.
 4. The composition of claim of claim 1, wherein the ratioof the Lactobacillus of step(b) is 1:1:1.
 5. The composition of claim 1,wherein the composition has a moisture content of less than about 5%;and a final bacterial concentration of about between 10⁵-10¹¹ colonyforming units (CFU) per gram of the composition.
 6. The composition ofclaim 1, further comprising an inert carrier.
 7. The composition ofclaim 6, wherein the inert carrier is dextrose monohydrate.
 8. Thecomposition of claim 7, wherein the dextrose monohydrate is at aconcentration of about between 75-95% (w/w).
 9. The composition of claim1, further comprising an organic emulsifier.
 10. The composition ofclaim 9, wherein the organic emulsifier is at a concentration of aboutbetween 2 to 5% (w/w).
 11. The composition of claim 9 wherein in theorganic emulsifier is soy lecithin.
 12. The composition of claim 1,comprising Bacillus of step (a) and the Bacillus of step (c).
 13. Thecomposition of claim 1, comprising Bacillus of step (a) and theLactobacillus of step (b).
 14. A method of treating wastewatercomprising contacting the wastewater with the composition of claim 13.15. The method of claim 14, wherein the wastewater is municipal sewage,residential septic, or industrial wastewater.
 16. The method of claim15, wherein the industrial wastewater comprises food, fats, oils,grease, brewery, agriculture, or commodity waste.
 17. The method ofclaim 15, wherein treating wastewater includes decreasing biologicaloxygen demand (BOD), total suspended solids (TSS) total kjeldahllnitrogen (TKN) and fats, oils and grease (FOG) in wastewater.
 18. Amethod of treating swimming pool water comprising contacting the waterwith the composition of claim
 13. 19. The method of claim 18, whereinthe water is contacted by contacting a swimming pool filtration unitwith the composition.
 20. The method of claim 18 wherein the compositionis imbedded in a solid support.
 21. A method of cleaning artificialturf, comprising, contacting the artificial turf with the composition ofclaim
 13. 22. A method of remediating wastewater from fruit or vegetablewashing comprising contacting the waste water with the composition ofclaim
 12. 23. A method of removing organic matter from the surfaces offruits and vegetables, comprising contacting the fruit or vegetable withthe composition of claim
 12. 24. The method of claim 23, wherein themethod improves the shelf life and/or appearance, of the fruit orvegetables.
 25. The method of claim 23, wherein the fruit is banana. 26.A method of manufacturing a composition comprising a mixture of bacteriacomprising Bacillus and Lactobacillus, wherein the method comprises thefollowing steps: a) individually aerobically fermenting each Bacillusorganism; b) individually anaerobically fermenting each Lactobacillusorganism; c) harvesting each Bacillus and Lactobacillus organism; d)drying the harvested organisms; e) grinding the dried organisms toproduce a powder f) combining each of the Bacillus powders to produce aBacillus mixture; g) combining each of the Lactobacillus powders inequal amounts to produce a Lactobacillus mixture; and h) combining theBacillus mixture and the Lactobacillus mixture at a ratio of between1:10 to 10:1.
 27. The method of claim 25, wherein the Bacillus comprisesBacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis,and Bacillus pumilus.
 28. The method of claim 25, wherein theLactobacillus comprises Pediococcus acidilactici, Pediococcuspentosaceus and Lactobacillus plantarum, Bacillus meagerium, Bacilluscoagulans, and Paenibacillus polymyxa.
 29. The method of claim 25,wherein the mixture has the following characteristics: a) a moisturecontent of less than about 5%; and b) a final bacterial concentration ofbetween about 10⁵-10¹¹ colony forming units (CFU) per gram of thecomposition.
 30. A method of manufacturing a composition comprising amixture of bacteria comprising Bacillus organisms, wherein the methodcomprises the following steps: a) individually aerobically fermentingBacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis,Bacillus pumilus, Bacillus meagerium, Bacillus coagulans, andPaenibacillus polymyxa; b) harvesting each organism; c) drying theharvested organisms; d) grinding the dried organisms to produce apowder; and e) combining each of the Bacillus powders.
 31. The method ofclaim 30, wherein the mixture has the following characteristics: a) amoisture content of less than about 5%; and b) a final bacterialconcentration of between about 10⁵-10¹¹ colony forming units (CFU) pergram of the composition.